Micro-organisms, including viruses, find many applications throughout the broad fields of biotechnology. They are involved in medicine, agriculture, industrial production processes (including notably the oil and brewing industries) and bioremediation. Many useful applications and functions have been identified and developed for such biological agents. However, often the development or enhancement of their activities is limited by their precise properties, restricting their ability to fulfil tasks that are theoretically possible but practically beyond their scope. In this situation, which is quite commonly encountered, it would often be desirable to re-engineer the properties of the virus or micro-organism to endow it with properties more appropriate for its required purpose.
Thus, biological insecticides for example such as baculoviruses may be restricted in their usefulness through inappropriate target specificity and adverse survival characteristics in the environment; sulphur metabolising bacteria may be limited in their useful application in the petrochemical industry through inadequate patterns of dispersion and distribution; and in the context of human or veterinary gene therapy, viruses intended to mediate delivery of therapeutic genes may be limited in their usefulness through inefficiency of transgene expression in target tissues.
The field of somatic cell gene therapy has attracted major interest in recent years because it promises to improve treatment for many different types of disease, including both genetic diseases (e.g. cystic fibrosis, muscular dystrophy, enzyme deficiencies) and diseases resulting from age- or damage-related physiological deterioration (cancer, heart disease, mature onset diabetics). However, although the field has seen rapid and extensive development, including initiation of over 100 clinical trials, instances of clear therapeutic benefit to patients are very few. One antisense technology has recently been licensed for human use, but no gene therapy strategies have as yet fulfilled their original promise and none are likely to be approved for routine clinical application in the foreseeable future.
The reasons for lack of therapeutic efficacy partly reflect the patient population (most patients enrolled for these experimental treatments are already quite sick so that even an effective treatment might show little therapeutic benefit) but primarily reflect the inadequate levels, duration and distribution of expression of therapeutic genes achieved. In short, the successful application of sophisticated treatment strategies is limited by inadequate vectors for gene delivery and expression.
Two main types of vectors for use in gene therapy applications have been explored so far—non-viral (usually based on cationic liposomes) and viral (usually retroviruses, adenoviruses, latterly adeno-associated viruses (aav) and lentiviruses).
Viruses are the obvious choice as vectors for gene delivery since this is essentially their sole function in nature. Consequently viruses have seen considerable use in gene therapy to date, forming the majority of vectors employed in clinical studies. The main feature of adenoviruses which limits their successful application is their immunogenicity. Although they are professional pathogens, evolved over millions of years as highly efficient gene delivery vectors, their hosts have similarly developed very effective protection mechanisms. Serum and ascites fluid from cancer patients contain antibodies that can completely prevent viral infection in vitro even at high dilution. Typical human protocols involving adenovirus lead to significant inflammatory responses, as well as inefficient infection of target cells.
Although the non-viral systems have a much better safety record, and are easier to produce in large quantities, they have low specific transfection activity and efficiency of gene expression in target tissues has been a major problem.
Another major limitation to successful application of presently available vectors for treatment of disease is a requirement for their administration directly to the site of disease, either by direct application or by intra-arterial administration. No vectors are capable of targeting to specific cells following intravenous injection. Cationic lipid systems occlude the first capillary bed they encounter, the pulmonary bed, while adenoviruses/retroviruses are rapidly taken up by the liver and (in animal studies) mediate local toxicities. Although local administration can be feasible for treatment of certain diseases (e.g. bronchial epithelial cystic fibrosis), other diseases have a more widespread distribution (notable clinical cancer and atherosclerosis) and intravenous targeted gene delivery is crucial to embrace the possibility of successful gene therapy.
One approach disclosed in WO 98/44143 for facilitating clinical use of viruses has been to modify the surface of the viruses with a mono-functional polymer such as poly(ethylene-glycol) (PEG). This can lead to significantly decreased neutralisation of infection by serum antibodies. This approach retains normal receptor-binding and infection in respect of target cells (via the CAR receptor for adenovirus), but presents a problem in that it does not facilitate targeting of the virus to selected receptors to gain useful and therapeutically-relevant tropisms.
WO 98/19710 discloses the use of multivalent polymers to coat cationic complexes of nucleic acid material to act as a-carrier vehicle for delivery of the nucleic acid material in biological systems. However, there is no disclosure or suggestion that the polymers would be useful to coat viruses or other micro-organisms.
One object of the present invention is to be able to target the virus or micro-organism to allow completely flexible tropism and modification of biological or physical properties.
Other objects and features of the invention will become apparent from the following more detailed description and Examples.